Bottom Line:
Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

ABSTRACTZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

Mentions:
A question naturally arose as to whether PDZ1 domains of ZO-1/ZO-2/ZO-3 interact with the cytoplasmic domain of claudins in epithelial cells. To evaluate this point, we constructed expression vectors for green fluorescent protein (GFP)-fusion proteins with PDZ1, PDZ2, or PDZ3 domains of ZO-1 and introduced them into cultured MDCK cells (Fig. 10, a–f). For an unknown reason, all these fusion proteins were concentrated in the nucleus. In addition to the nuclear staining, PDZ1-GFP and PDZ2-GFP were clearly recruited to and concentrated at claudin-1–positive TJs (Fig. 10, a–d). Similarly to L transfectants, these findings can be interpreted as indicating that PDZ1-GFP and PDZ2-GFP are recruited by the direct association with endogenous claudins and ZO-2/ZO-3, respectively. In epithelial cells, PDZ3-GFP also appeared to be concentrated at TJs, although very faintly as compared with PDZ1-GFP and PDZ2-GFP (Fig. 10e and Fig. f), probably due to unidentified binding partners for PDZ3 domain of ZO-1 localized at TJs in epithelial cells. GFP-fusion proteins with PDZ1 (Fig. 10, g–j) or PDZ2 (data not shown) domains of ZO-2/ZO-3 also showed significant concentration at claudin-1–positive TJs in MDCK cells.

Mentions:
A question naturally arose as to whether PDZ1 domains of ZO-1/ZO-2/ZO-3 interact with the cytoplasmic domain of claudins in epithelial cells. To evaluate this point, we constructed expression vectors for green fluorescent protein (GFP)-fusion proteins with PDZ1, PDZ2, or PDZ3 domains of ZO-1 and introduced them into cultured MDCK cells (Fig. 10, a–f). For an unknown reason, all these fusion proteins were concentrated in the nucleus. In addition to the nuclear staining, PDZ1-GFP and PDZ2-GFP were clearly recruited to and concentrated at claudin-1–positive TJs (Fig. 10, a–d). Similarly to L transfectants, these findings can be interpreted as indicating that PDZ1-GFP and PDZ2-GFP are recruited by the direct association with endogenous claudins and ZO-2/ZO-3, respectively. In epithelial cells, PDZ3-GFP also appeared to be concentrated at TJs, although very faintly as compared with PDZ1-GFP and PDZ2-GFP (Fig. 10e and Fig. f), probably due to unidentified binding partners for PDZ3 domain of ZO-1 localized at TJs in epithelial cells. GFP-fusion proteins with PDZ1 (Fig. 10, g–j) or PDZ2 (data not shown) domains of ZO-2/ZO-3 also showed significant concentration at claudin-1–positive TJs in MDCK cells.

Bottom Line:
Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited.When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1.Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions.

ABSTRACTZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.